Low-molecular weight peptides inhibiting ion channel activity

ABSTRACT

It is intended to provide novel polypeptides which specifically inhibit the activity of a mechano-sensitive channel; and mechano-sensitive channel inhibitors or remedies for atrial fibrillation containing these polypeptides or salts thereof. The above objects can be achieved by using polypeptides having amino acid sequences represented by SEQ ID NO: 1 (TVP003), SEQ ID NO:2 (TVP004) and SEQ ID NO:3 (TVP005), salts of these polypeptides, and mechano-sensitive channel inhibitors or remedies for atrial fibrillation containing the same.

This application is a divisional application of U.S. Ser. No.10/550,102, filed Sep. 21, 2005, which is a national stage ofInternational Application No. PCT/JP2004/004190 filed on Mar. 25, 2004,which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a polypeptide that inhibits theactivity of a mechano-sensitive channel, and relates to amechano-sensitive channel inhibitor and remedy for atrial fibrillationcomprising the polypeptide. More specifically, the present inventiondetermines the pharmacophore that acts upon the mechano-sensitivechannel based on the sequence of a natural peptide from spider venom(GsMTx-4), and it relates to novel polypeptides designed to compose thepharmacophore which are moreover useful for atrial fibrillationtreatment.

BACKGROUND OF ART

Atrial-fibrillation is-a type of arrhythmia, of which the morbidityprevalence rate increases with advanced age. Atrial fibrillation is aheart disease observed in 3% of the elderly (over 65 years old). Whenatrial fibrillation becomes chronic, it forms a thrombosis and inducescerebral thrombosis, and therefore it is currently thought that atrialfibrillation is the main disease factor of serious cases of cerebralapoplexy. Thus, considering the frequency and seriousness ofcomplications such as cerebral infarction, atrial fibrillation has cometo be regarded in recent years as one kind of lethal arrhythmia (J.Nippon. Med. Sch. 2002, 69(3)). Heretofore, a remedy that completelycures atrial fibrillation had not been obtained, and so it had beenassumed that medication for atrial fibrillation, particularly chronicatrial fibrillation, had its limits (J. Nippon. Med. Sch. 2002, 69(3)).

Atrial fibrillation is believed to be caused in part by themalfunctioning of an ion channel in the myocardium. Meanwhile, a naturalpeptide from spider venom (GsMTx-4: SEQ ID NO:4) is known to inhibit theactivity of a mechano-sensitive channel (Stretch-Activated Channel: SAC)(see for example Thomas M. Suchyna et al., Identification of a PeptideToxin from Grammostola Spatulata Spider Venom that BlocksCation-selective Stretch-activated Channels, J. Gen. Physiol., Vol. 115,pp583-598 (2000) (non-patent document 1)). In the said document, it isdescribed that in peptides composing toxins derived from venoms fromterrestrial and aquatic animals, an ICK (Inhibitor Cysteine Knot) motifwith six cysteines is commonly observed (non-patent document 1, p.590,right column, 11.7 to 3 from bottom, and FIG. 30). The said documentalso suggests that GsMTx-4 has an ICK motif with a basic structuredefined by three cysteine pairs (C₁-C₄, C₂-C₅ and C₃-C₆) (non-patentdocument 1, p.595, left column, 1.7 to 1.11 from bottom, column ‘Thestructure of GsMTx-4’).

Furthermore, methods for extracting and purifying GsMTx-4, methods fortreating cerebral arrhythmia with the said GsMTx-4 and so forth havebeen suggested (see for example Bode et al., Nature, Vol.409, pp35-36(2001) (non-patent document 2), US Patent Application PublishedDescription No.2002/0077286 (patent document 1)). Further, the structureof GsMTx-4 has been known from results obtained in a solution using NMR(see Robert et al., J. Biol. Chem. Vol.37, pp3443-3445, 2002.(non-patent document 3)). Despite such findings, a remedy for atrialfibrillation using the peptide derived from spider venom (GsMTx-4) hadnot been developed.

REFERENCES

Patent document 1: US Patent Application Published DescriptionNo.2002/0077286;

Non-patent document 1: Thomas M. Suchyna et.al., Identification of aPeptide Toxin from Grammostola Spatulata Spider Venom that BlocksCation-selective Stretch-activated Channels, J. Gen. Physiol., Vol.115,pp583-598 (2000);

Non-patent document 2: Bode et al., Nature, Vol.409, pp35-36 (2001);

Non-patent document 3: Robert et al., J. Biol. Chem. Vol.37, pp3443-3445(2002).

The object of the present invention is to identify the pharmacophore(the minimum space structure needed for activation) of GsMTx-4, todesign novel polypeptides that specifically inhibit the activity of amechano-sensitive channel based on the pharmacophore information, and toprovide remedies for atrial fibrillation comprising such polypeptides.

SUMMARY OF INVENTION

The above objects are achieved by the following inventions.

[1] In a first aspect of the present invention, it involves apolypeptide or salts thereof’ consisting of an amino acid sequencerepresented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. Thesepolypeptides, as confirmed in the embodiment of this description, arepolypeptides that show mechano-sensitive channel inhibiting activity,and can be considered as polypeptides that compose the pharmacophore ofGsMTx-4. These polypeptides are useful for treatment of atrialfibrillation and such.[2] In a second aspect of the present invention, it involves apolypeptide or salts thereof comprising an amino acid sequencerepresented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.[3] In a third aspect of the present invention, it involves apolypeptide or salts thereof consisting of an amino acid sequencerepresented by SEQ ID NO:16 or SEQ ID NO:17. These polypeptides, asconfirmed in the embodiment of this description, are polypeptides thatshow mechano-sensitive channel inhibiting activity. These polypeptidesare useful for treatment of atrial fibrillation and such.[4] In a fourth aspect of the present invention, it involves apolypeptide or salts thereof consisting of an amino acid sequencerepresented by SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO:3 of which one ormore of the amino acids thereof have been deleted, substituted, insertedor added, but not comprising an amino acid sequence described in SEQ IDNO:4, and moreover showing mechano-sensitive channel inhibitingactivity.[5] In a fifth aspect of the present invention, it involves apolypeptide or salts thereof described in the above [4] as consisting ofan amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ IDNO:3 of which one or more of the amino acids thereof have been deleted,substituted, inserted or added, but not comprising an amino acidsequence described in SEQ ID NO:4, and moreover showingmechano-sensitive channel inhibiting activity, of which the saidpolypeptide comprises an amino acid sequence represented by SEQ ID NO:16or SEQ ID NO:17.[6] In a sixth aspect of the present invention, it involves apolynucleotide comprising a polynucleotide that encodes the polypeptidedescribed in the above [1], the above [3] or the above [4].[7] In a seventh aspect of the present invention, it involves arecombinant vector comprising the polynucleotide described in the above[6].[8] In a eighth aspect of the present invention, it involves atransformant transformed by the recombinant vector described in theabove [7].[9]0 In a ninth aspect of the present invention, it involves amechano-sensitive channel inhibitor comprising one or more of thepolypeptides or salts thereof described in one of the above [1] to [5].This inhibitor specifically inhibits the activity of a mechano-sensitivechannel and thus is useful for conducting researches onmechano-sensitive channels and such.[10] In a tenth aspect of the present invention, it involves a remedyfor atrial fibrillation comprising one or more of the polypeptides orsalts thereof described in one of the above [1] to [5]. Thesepolypeptides, as confirmed of their functions in the embodiment of thisdescription, show mechano-sensitive channel inhibiting activity.Therefore, this remedy can be used effectively in treating atrialfibrillation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the ten prospective multiple alignmentsshowing high homology to GsMTx-4

FIG. 2 is a representation of the alignment of 1QK6 and GsMTx-4.

FIG. 3 is a stereo view superimposing Huwentoxin-I and GsMTx-4.

FIG. 4 is a Cα trace of the model of the superimposed Huwentoxin-I andGsMTx-4. In FIG. 4, the thin lines indicate the template and the thicklines indicate GsMTx-4.

FIG. 5 is a representation of the vicinity of Arg20 considered to be theactivity center of Huwentoxin-I. In FIG. 5, the thin lines indicateHuwentoxin-I and the thick lines indicate GsMTx-4.

FIGS. 6( a) to 6(d) are representations of the surface structure of amodel with Huwentoxin-I (PDB code: 1QK6) as the template. In FIGS. 6( a)to 6(d), the left side indicates Huwentoxin-I, and the right sideindicates GsMTx-4. FIG. 6( a) is a view of the model from a hydrophobicpatch (approximately the same direction as the drawings above). FIG. 6(b) is a view of the model rotated +90° around the x-axis. FIG. 6( c) isa view of the model rotated +90° around the y-axis. FIG. 6( d) is a viewof the model rotated 180° around the y-axis.

FIG. 7 is a representation of the structure of GsMTx-4 and thestructures of the designed peptides.

FIGS. 8( a) and 8(b) are representations of the inhibiting activity ofTVP003. FIG. 8( a) is a representation of the single channel currentrecordings. FIG. 8( b) represents the channel's open probability (Po).

FIGS. 9( a) and 9(b) are representations of the inhibiting activity ofTVP004. FIG. 9( a) is a representation of the single channel currentrecordings. FIG. 9( b) represents the channel's open probability (Po).

FIGS. 10( a) and 10(b) are representations of the inhibiting activity ofTVP005. FIG. 10( a) is a representation of the single channel currentrecordings. FIG. 10( b) represents the channel's open probability (Po).

FIGS. 11( a) and 11(b) are representations of the inhibiting activity ofTVP017. FIG. 11( a) is a representation of the single channel currentrecordings. FIG. 11( b) represents the channel's open probability (Po).

FIGS. 12( a) and 12(b) are representations of the inhibition activity ofTVP019. FIG. 12( a) is a representation of the single channel currentrecordings. FIG. 12( b) represents the channel's open probability (Po).

FIGS. 13( a) to 13(c) are results of the study into the specificity ofthe active peptide to a mechano-sensitive channel. FIG. 13( a) is arepresentation of the single channel current recordings related to themyocardial SA channel of TVP003. FIG. 13( b) is a representation of thesingle channel current recordings related to the STREX-deletion-mutantof TVP0003. FIG. 13( c) represents the channel's open probability (Po).

BEST MODE FOR CARRYING OUT THE INVENTION (Polypeptides of the PresentInvention)

The Polypeptides of the present invention consist of a polypeptideconsisting of an amino acid sequence represented by SEQ ID NO:1, SEQ IDNO:2 or SEQ ID NO:3; a polypeptide comprising an amino acid sequencerepresented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; a polypeptideconsisting of an amino acid sequence represented by SEQ ID NO:16 or SEQID NO:17; a polypeptide consisting of an amino acid sequence representedby SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 of which one or more of theamino acids have been deleted, substituted, inserted or added, but notcomprising an amino acid sequence described in SEQ ID NO:4, and moreovershowing mechano-sensitive channel inhibiting activity (namely, thepolypeptides involved in embodiments 1 to 5 of the present invention).

Furthermore, the polypeptides of the present invention may have aC-terminus in the form of a carboxyl group (—COOH), a carboxylate(—COO⁻), an amide (—CONH₂), an ester (—COOR) or the like.

The polypeptides of the present invention include polypeptides whereinthe amino group at the methionine residue of the N-terminus is protectedwith a protecting group. The polypeptides of the present inventioninclude polypeptides wherein the N-terminal is cleaved in vivo and theG1n thus formed is pyroglutaminated. The polypeptides of the presentinvention include polypeptides wherein a substituent on a side chain isprotected by an appropriate protecting group. The polypeptides of thepresent invention include synthetic polypeptides such as the so-calledglycoproteins having conjugated sugar chains.

The salts of the polypeptides of the present invention may be salts inthe form of physiologically acceptable salts with acids or bases,preferably in the form of physiologically acceptable acid additionsalts. Examples of such salts include salts with inorganic acids (e.g.hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid),and salts with organic acids (e.g. acetic acid, formic acid, propionicacid, fumaric acid, maleic acid, succinic acid, tartaric acid, citricacid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid,benzensulfonic acid).

(Composition of the Polypeptides of the Present Invention)

The polypeptides of the present invention may be prepared by chemicalsynthesis, or may be manufactured using recombinant DNA technology. Toprepare the polypeptides of the present invention by chemical synthesis,the publicly known methods may be used, for example, the peptide of thepresent invention can be obtained by methods using azide, acid chloride,acid anhydride, compound acid anhydride, DCC, activated ester,Woodward's reagent K, carbonylimidazole, deoxidization, DCC/HONB, BOPreagent (see for example Bozanszky, M and M. A. Ondetti, PeptideSynthesis, Interscience Publishers, New York (1966); Schroeder andLuebke, The Peptide, Academic Press, New York (1 965); F. M. Finn and K.Hofmann, The Proteins Vol.2, H. Nenrath, R. L. Hill ed., Academic PressInc., New York (1976); Nobuo Izumiya et al., Peptide Gosei no Kiso toJikken (Basics and experiments of peptide synthesis), Maruzen Co.(1985); Haruaki Yajima and Shunpei Sakakibara et al., Seikagaku JikkenKoza (Biochemical Experiment) 1, Japanese Biochemical Society ed., TokyoKagaku Dojin Co. (1977); Toshiya Kimura, Zoku Seikagaku Jikken Koza(Sequel to Biochemical Experiment) 2, Japanese Biochemical Society ed.,Tokyo Kagaku Dojin Co. (1987)). Furthermore, the peptide of the presentinvention can be prepared by chemical synthesis using an automatedpeptide synthesizer (e.g. PE Applied Bio Systems Co.).

Further, following the completion of reaction, the polypeptides of thepresent invention can be purified and separated by publicly knownpurification methods. For example, the polypeptide of the presentinvention can be purified and separated by a combination of solventextraction, distillation, column chromatography, liquid chromatography,recrystallization and the like. Where the peptide of the presentinvention obtained by the above methods is in a free form, publiclyknown methods can be used to convert it into a salt form, and on theother hand, where the peptide is obtained in a salt form, publicly knownmethods can be used to convert it into a free form.

(Polynucleotide Encoding the Polypeptide)

The polynucleotide encoding the polypeptide of the present invention maybe any polynucleotide so long as it contains the base sequence (DNA orRNA, preferably DNA) encoding the polypeptide of the present invention.For example, the polynucleotide may be the DNA or RNA such as mRNAencoding the polypeptide of the present invention, and it can either bedouble stranded or single stranded. When double stranded, it may be adouble stranded DNA, a double stranded RNA or a hybrid of DNA and RNA.When single stranded, it may either be a sense strand (namely, a codingstrand) or an anti-sense strand (namely, a non-coding strand).

Using the polynucleotide encoding the polypeptide of the presentinvention, mRNA of the polypeptide of the present invention can beassayed, for example, according to the publicly known method describedin the special issue of Jikken Igaku (Experimental Medicine), Shin PCRto Sono Oyo (Novel PCR and. its application) 15(7), 1997, or accordingto a similar method.

The DNA encoding the polypeptide of the present invention includegenomic DNA, genomic DNA library, cDNA derived from the cells or tissuesdescribed above, cDNA library derived from the cells and tissuesdescribed above, and synthetic DNA, of which any one thereof can beemployed. Examples of the vector used for the library includebacteriophage, plasmid, cosmid and phagemide, of which any one thereofcan be employed. Further, the DNA can be directly amplified by ReverseTranscriptase Polymerase Chain Reaction (hereinafter abbreviated asRT-PCR) employing a total RNA or a mRNA fraction prepared from the cellsor tissues described above.

For cloning the DNA encoding the polypeptide of the present invention,there is the method of amplifying by PCR using synthetic DNA primerscomprising a part of the base sequence of the DNA encoding thepolypeptide of the present invention. The cloning of the DNA encodingthe polypeptide of the present invention can also be performed byselecting the DNA inserted into an appropriate vector by hybridizationwith a labeled DNA fragment or synthetic DNA that encodes a part of theregion or the entire region of the polypeptide of the present invention.Hybridization can be carried out, for example, according to the methoddescribed in Molecular Cloning 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989). When using commercially available library,hybridization can be carried out according to the method described inthe attached instructions.

Substitution of the base sequence of the DNA can be carried out by PCRor by using publicly known kits, for example, Mutan™-super Express Km(Takara Shuzo Co.) and Mutan™-K (Takara Shuzo Co.) according to publiclyknown methods such as ODA-LA PCR, Gapped duplex method and Kunkelmethod, or according to similar methods.

The cloned DNA encoding the polypeptides can be used as it is, or ifdesired, used after digesting with a restriction enzyme or after addinga linker thereto. Such DNA can contain ATG as translation initiationcodon at the 5′ end thereof and can contain TAA, TGA or TAG astranslation termination codon at the 3′ end thereof. These translationinitiation codon and translation termination codon can be added by usingan appropriate synthetic DNA adapter.

The expression vector of the polypeptide of the present invention can bemanufactured, for example, by excising a desired DNA fragment from theDNA encoding the polypeptide of the present invention and thenlitigating the DNA fragment downstream of a promoter in an appropriateexpression vector.

Examples of the vector include plasmids derived from a Escherichia coli(e.g., pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), plasmids derivedfrom Bacillus subtlis (e.g., pUB110, pTP5, pC194), plasmids derived fromyeast (e.g., pSH19, pSH15), bacteriophages such as _(λ) phage, animalviruses such as retrovirus, vaccinia virus and baculovirus, as well aspA1-11, pXT1, pRc/CMV, pRc/RSV and pcDNAI/Neo.

The promoter employed in the present invention can be any promoter solong as it is an appropriate promoter matching the host used for geneexpression. For example, when using animal cells as the host, SRαpromoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter orsuch can be utilized.

Of these, it is preferable to use CMV promoter, SRα promoter or thelike. When using bacteria of the genus Escherichia as the host, thepreferred promoter is trp promoter, 1ac promoter, recA promoter,_(λ)P_(L) promoter, 1pp promoter or the like, when using bacteria of thegenus Bacillus as the host, the preferred promoter is SPO1 promoter,SPO2 promoter, penP promoter or the like, when using yeast as the host,the preferred promoter is PHO5 promoter, PGK promoter, GAP promoter, ADHpromoter or the like. When using insect cells as the host, it ispreferable to use polyhedron promoter, P10 promoter or the like.

For the expression vector, in addition to the above examples, thosecomprising an enhancer, a splicing signal, a poly A addition signal, aselection marker, an SV40 replication origin (hereafter may beabbreviated as SV40Ori) or such can be used if so desired. Examples ofthe selection marker include dihydrofolate reductase (hereinafter may beabbreviated as dhfr) gene (methotrexate (MTX) resistance), amplicillonresistant gene (hereinafter may be abbreviated as Amp^(r)), neomycinresistant gene (hereinafter may be abbreviated as Neo^(r), G418resistance) and the like. In particular, when using dhfr gene as theselection marker by employing CHO (dhfr⁻) cells, the desired gene may beselected using a medium not comprising thymidine.

When necessary, a signal sequence matching the host is added to theN-terminus of the polypeptide of the present invention. When usingbacteria of the genus Escherichia as the host, PhoA signal sequence,OmpA signal sequence or such can be utilized, when using bacteria of thegenus Bacillus as the host, α-amylase signal sequence, subtilisin signalsequence or such can be utilized, when using yeast as the host, MFαsignal sequence, SUC2 signal sequence or such can be utilized, and whenusing animal cells as the host, insulin signal sequence, α-interferonsignal sequence, antibody molecule signal sequence or such can beutilized.

Using the vector comprising the DNA encoding the polypeptide of thepresent invention thus composed, transformants can be manufactured.

Examples of the host include bacteria of the genus Escherichia, bacteriaof the genus Bacillus, yeast, insect cells, insect and animal cells.

Specific examples of the bacteria of the genus Escherichia includeEscherichia coli K12·DH1 (Proc. Natl. Acad. Sci. USA, 60, 160 (1968)),JM103 (Nucleic Acids Research, 9, 309 (1981)), JA221 (Journal ofMolecular Biology, 120, 517 (1978)), HB101 (Journal of MolecularBiology, 41, 459 (1969)), C600 (Genetics, 39, 440 (1954)), DH5α (Inoue,H., Nojima, H. and Okayama, H., Gene, 96, 23-28 (1990)), and DH10B(Proc. Natl. Acad. Sci. USA, 87, 4645-4649 (1990)).

Examples of the bacteria of the genus Bacillus include Bacillus subtilisMi114 (Gene, 24, 255 (1983)), and 207-21 (Journal of Biochemistry, 95,87 (1984)).

Examples of yeast include Saccharomyces cerevisiae AH22, AH22R—,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,and Pichia pastoris.

For insect cells, where the virus is AcNPV, Spodoptera frugiperda cells(Sf cells), MG1 cells derived from the mid-intestine of Trichoplusia ni,HighFive™ cells derived from the egg of Trichoplusoa ni, cells derivedfrom Mamestra brassicae, cells derived from Estigmena acrea and the likecan be used. Where the virus is BmNPV, Bombyx mori N cells (BmN cells)and the like can be used. Examples of the Sf cells thereof include Sf9cells (ATCC CRL1711), and Sf21 cells (see Vaughn, J. L. et al., In Vivo,13, 213-217 (1977) for above).

For insects, the larva of silkworms can be used (Maeda et al., Nature,315, 592 (1985)).

Examples of animal cells include monkey cells COS-7, Vero, Chinesehamster cells CHO (hereinafter abbreviated as CHO cells), dhfr genedeficient Chinese hamster cells CHO (hereinafter abbreviated asCHO(dhfr⁻) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, ratGH3, and human FL cells.

Bacteria of the genus Escherichia can be transformed according to themethod described, for example, in Proc. Natl. Acad. Sci. USA, 69, 2110(1972) or in Gene, 17 107 (1982).

Bacteria of the genus Bacillus can be transformed according to themethod described, for example, in Molecular & General Genetics, 168, 111(1979).

Yeast can be transformed according to the method described, for example,in Methods in Enzymology, 194, 182-187 (1991) or in Proc. Natl. Acad.Sci. USA, 75, 1929 (1978)

Insect cells and insects can be transformed according to the methoddescribed, for example, in Bio/Technology, 6, 47-55 (1988)

Animal cells can be transformed according to the method described, forexample, in Saibo Kogaku (Cell Engineering) extra issue 8, Shin SaiboKogaku Jikken Protocol (New Cell Engineering Experimental Protocol),263-267, Shujunsha (1995) or in Virology, 52, 456 (1973).

Thus, a transformant transformed with the expression vector comprisingthe DNA encoding the polypeptide of the present invention can beobtained.

In cultivating the transformant having bacteria of the genus Escherichiaor bacteria of the genus Bacillus as the host, the appropriate mediumused for cultivation is a liquid medium wherein carbon sources, nitrogensources, inorganic substances and such required for the growth of thesaid transformant are contained. Carbon sources include glucose,dextrin, soluble starch, sucrose and the like, nitrogen sources includeinorganic or organic matter such as ammonium salts, nitrate salts, cornsteep liquor, peptone, casein, meat extract, soybean cake, potatoextract and the like, and inorganic substances include calcium chloride,sodium dihydrogenphosphate, magnesium chloride and the like. Inaddition, yeast extract, vitamins, growth promoting factors and such canbe added. The pH of the medium is preferably about 5 to 8.

The preferred medium for cultivating bacteria of the genus Escherichiais M9 medium comprising glucose and Casamino acids (Miller, Journal ofExperiments in Molecular Genetics, 431-433, Cold Spring HarborLaboratory, New York (1972)) or the like. When necessary, chemicals suchas 3β-indolylacrylic acids can be added therein to make the promoterfunction efficiently.

When using bacteria of the genus Escherichia as the host, cultivation isgenerally carried out at about 15 to 43° C. for about 3 to 24 hours, andaeration or agitation can be conducted when necessary.

When using bacteria of the genus Bacillus as the host, cultivation isgenerally carried out at about 30 to 40° C. for about 6 to 24 hours, andaeration or agitation can be conducted when necessary.

When cultivating a transformant with yeast as the host, the medium to beused is, for example, Burkholder's minimal medium (Bostian, K. L. etal., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)), or SD mediumcomprising 0.5% Casamino acids (Bitter, G A. et al., Proc. Natl. Acad.Sci. USA, 81, 5330 (1984)). Preferably, pH of the medium is adjusted toabout 5 to 8. Cultivation is generally carried out at about 20 to 35° C.for about 24 to 72 hours, and aeration or agitation is conducted whennecessary.

When cultivating a transformant with insect cells or insects as thehost, the medium to be used is, for example, Grace's Insect Medium(Grace, T. C. C., Nature, 195,788 (1962)) to which is added appropriateamounts of additives such as immobilized 10% calf serum. Preferably, pHof the medium is adjusted to about 6.2 to 6.4. Cultivation is generallycarried out at about 27° C. for about 3 to 5 days, and aeration oragitation is conducted when necessary.

When cultivating a transformant with animal cells as the host, themedium to be used is, for example, MEM medium comprising about 5 to 20%of fetal calf serum (Science, 122, 501 (1952)), DMEM medium (Virology,8, 396 (1959)), RPMI 1640 medium (Journal of the American MedicalAssociation, 199, 519 (1967)), or 199 medium (Proceeding of the Societyfor the Biological Medicine, 73, 1 (1950)) Preferably, pH should beabout 6 to 8. Cultivation is carried out at about 30 to 40° C. for about15 to 60 hours, and aeration or agitation is conducted when necessary.

In the foregoing manner, the polypeptide of the present invention can beproduced in the transformant intracellularly, in cell membranes, orextracellularly.

The polypeptide of the present invention can be separated and purifiedfrom the above culture medium using the methods described below.

When extracting the polypeptide of the present invention from thecultured bacteria or cells, appropriate methods are used in whichfollowing cultivation, bacteria or cells are collected by publicly knownmethods and suspended in an appropriate buffer, the bacteria or cellsare then disrupted by ultrasound, lysozyme and/or freeze-thawing, afterwhich the crude extract of the polypeptide is obtained throughcentrifugation or filtration. The buffer may contain a protein modifiersuch as urea or guanidine hydrochloride, or a surfactant such as TritonX-100™. Where the polypeptide is secreted into the culture broth,following the completion of cultivation, the bacteria or cells areseparated from the supernatant using a publicly known method, and thesupernatant is collected.

The polypeptide contained in the cultured supernatant or the extractthus obtained can be purified by appropriately combining publicly knownseparation and purification methods. Such publicly known separation andpurification methods include methods that make use of solubility such assalting out and solvent precipitation, methods that make use ofdifference in molecular weight such as dialysis, ultrafiltration, gelfiltration and SDS-polyacrylamide gel electrophoresis, methods that makeuse of difference in electric charge such as ion exchangechromatography; methods that make use of the difference in specificaffinity such as affinity chromatography, methods that make use ofdifference in hydrophobicity such as reverse phase high performanceliquid chromatography, and methods that make use of difference inisoelectric point such as isoelectrofusing electrophoresis.

Where the polypeptide thus obtained is in a free form, publicly knownmethods or similar methods can be used to convert it into a salt form,and on the other hand, where it is obtained in a salt form, publiclyknown methods or similar methods can be used to convert it into a freeform or into another salt form.

Further, the polypeptide produced by the recombinant can be optionallymodified and parts of the polypeptide can be removed by activating anappropriate protein modifying enzyme before or after purification.Examples of the protein-modifying enzyme include trypsin, chymotrypsin,arginyl endopeptidase, protein kinase and glycosidase.

The activity of the polypeptide of the present invention or saltsthereof thus generated can be measured by a binding experiment to alabeled ligand, an enzyme immunoassay using a specific antibody, orsuch.

(Mechano-Sensitive Channel Inhibitors)

A mechano-sensitive channel inhibitor contains, for example, one or moreof either the polypeptides of the present invention or salts thereof(hereinafter may be described as polypeptides of the present invention).The polypeptides of the present invention can be utilized asmechano-sensitive channel inhibitors. The polypeptides of the presentinvention are easy to handle, and as shown in the embodiments below,display high inhibiting activity.

(Remedies for Atrial Fibrillation)

A remedy for atrial fibrillation contains, for example, one or more ofeither the polypeptides of the present invention or salts thereof. Inother words, the present invention can provide pharmaceuticals andpharmaceutical compositions as well. Pharmaceutical compositions are,for example, those that contain the polypeptide of the present inventionor salts thereof and a pharmaceutically acceptable carrier.

The remedy for atrial fibrillation comprising the polypeptide of thepresent invention can be administered parenterally, for example, intothe blood vessel of the heart in the form of an injection, or can beused orally, for example, in the form of tablets or capsules.Formulations for injection can be provided in ampules comprising a unitdosage or in containers comprising multiple dosages. Moreover, thepreparations can be administered not only to humans but also to otherwarm-blooded animals. The formulations can be prepared using publiclyknown preparation methods.

The various preparations can be manufactured using conventional methodsby appropriately selecting generally used formulations such as anexcipient, a swelling agent, a binder, a moistening agent, a disruptingagent, a lubricant, a surface-active agent, a dispersing agent, abuffer, a preservative, a solubilizing agent, an antiseptic, asweetening and flavoring agent, a soothing agent, a stabilizing agent,and an isotonic agent. The various agents described above may alsocontain pharmaceutically acceptable carriers or additives. Such carriersor additives include water, pharmaceutically acceptable organicsolvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone,carboxyvinylpolymer, alginic acid sodium, water-soluble dextran,carboxymethyl starch sodium, pectin, Xanthan gum, gum arabic, casein,gelatin, agar, glycerin, propylene glycol, polyethylene glycol,Vaseline, paraffin, stearate alcohol, stearic acid, human serum albumin,mannitol, sorbitol, and lactose. The additives to be used are selectedappropriately or in combination from those described above according tothe formulations of the present invention.

The polypeptide of the present invention consisting of the activatedcomponent in the forms described above is contained therein, forexample, 0.01 to 100% by weight, preferably 0.1 to 90% by weight, morepreferably 1 to 50% by weight.

Concerning the dosage of the polypeptide of the present invention, whenadministered parenterally, the amount of one dosage differs depending onthe subject of administration, symptoms and route of administration; andso, if administered in the form of injection to a patient (weighing 60kg) with atrial fibrillation, for example, the daily dosage is generallyabout 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferablyabout 0.1 to 10 mg. When administered orally to an atrial fibrillationpatient (weighing 60 kg), for example, the daily dosage is about 0.1 to100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20mg. The remedy for atrial fibrillation of the present invention ispreferably administered once to several times a day for a duration ofmore than one day.

(Identification of the Pharmacophore)

In designing the polypeptide of the present invention, the region of thepeptide from spider venom (GsMTx-4) consisting of the pharmacophore thatis the minimal unit necessary for activation has been estimated withgood precision based on a three-dimensional structure.

The pharmacophore can be identified for example in the manner describedhereinafter. First, a precise structure estimation of the peptide fromspider venom is conducted according to the homology modeling methodusing a similar peptide with a known three-dimensional structure as thetemplate. Based on the structure thus obtained, a function analysis ofthe peptide with a modified activated part is performed and the targetpart for pharmaceutical design is narrowed down. A design that mimicsthe disulfide bond region of GsMTx-4 is created, and the peptide with astable structure is designed. GsMTx-4 is known to have athree-dimensional structure of relatively low flexibility that comprisesthree disulfide bonds, and so the pharmacophore needed for activation isidentified by designing several cyclic peptides comprising polar aminoacid residues generally often involved in the binding process.

(Measurement of the Activity)

The activity evaluation method of the peptide of the present inventioncan be carried out using the publicly known activity evaluation method,preferably using the single channel current recording method employingthe patch clamp method described in embodiment 1.

Embodiment 1 Example 1: Search for a Similar Peptide with a KnownStructure Showing High Homology to the Sequence of the Peptide from theSpider Venom (GsMTx-4)

A structure with high homology to the amino acid sequence of GsMTx-4represented by SEQ ID NO:4 was searched among the spider venoms from PDB(Protein Data Bank). Consequently, 10 candidates with high homology toGsMTx-4 were found. The results of the multiple alignment of thosecandidates are shown in FIG. 1 below.

Of the sequences shown in FIG. 1, 1QK6 (Huwentoxin-I: SEQ ID NO:12)comprising the same cysteine residues, and moreover having almost thesame length between the cysteine residues with no insertion or deletion(GsMTx-4 is one residue longer) was selected as the template to narrowdown the pharmacophore using the homology modeling method.

Example 2: Estimation of the Three Dimensional Structure of the Peptideof Spider Venom (GsMTx-4).

Homology modeling was carried out using the template peptide 1QK6.First, alignment of 1QK6 and GsMTx-4 was performed. The result is shownin FIG. 2. Next, the model structure was constructed with programMODELLER. The superimposition of the constructed structure model ofGsMTx-4 and Huwentoxin-I used as the template is shown in FIG. 3 and inFIG. 4. FIG. 3 is a stereo view of the superimposed Huwentoxin-I andGsMTx-4. FIG. 4 is a Cα trace of the model of the superimposedHuwentoxin-I and GsMTx-4. In FIG. 4, the thin lines indicate thetemplate, and the thick lines indicate GxMTx-4.

The vicinity of Arg20 considered to be the activity center ofHuwentoxin-I is shown in FIG. 5. In FIG. 5, the thin lines indicateHuwentoxin-I, and the thick lines indicate GsMTx-4. Furthermore,comparisons of the surface structures of Huwentoxin-I and GsMTx-4 areshown in FIG. 6. In FIGS. 6( a) to 6(d, the left side showsHuwentoxin-I, and the right side shows GsMTx-4. FIG. 6( a) is a viewfrom a hydrophobic patch (approximately the same direction as thedrawings above). FIG. 6( b) is a view rotated +90° around the x-axis.FIG. 6( c) is a view rotated +90° around the y-axis. FIG. 6( d) is aview rotated 180° around the y-axis. FIG. 6( b) shows that the moleculesof the two peptides have quite different forms. The distributions of theresidues with isolable side chains also differ, and can be assumed to beconcerned with the determination of specificity.

Further, by comparing the structure of GsMTx-4 obtained in a solutionusing NMR disclosed in Robert et. al., J. Biol. Chem. Vol.37,pp3443-3445, 2002 (non-patent document 3 mentioned above) and thestructure of GsMTx-4 obtained in the present embodiment, the modelingstructure constructed in the present invention can be judged asreflecting the actual structure of GsMTx-4.

Example 3: Design of the Active Peptide and Identification of thePharmacophore Thereof

Based on the modeling structure of GsMTx-4 described above, the policyfor designing the peptide fragments was decided. The structure ofGsMTx-4 and the designed structure of the peptides are shown in FIG. 7.GsMTx-4 comprises four loops formed by three disulfide bonds asindicated in the sequence structure formula shown in FIG. 7. Therefore,five peptides TVP001 to TVP005 were designed to determine the loopcontributing to the inhibiting activity. In these peptides, cysteines atthe regions not composing the loops have been substituted with alanines.TVP011 (SEQ ID NO:14) comprises loops 1 and 2, TVP002 (SEQ ID NO:15)comprises loops 3 and 4, TVP003 (SEQ ID NO:1) comprises loop 2, TVP004(SEQ ID NO:2) comprises loop 3, and TVP005 (SEQ ID NO:3) comprises loops2 and 3.

(Bioassay of the Designed Peptides)

Activity assay of the peptides was carried out by the most reliablesingle channel current recording method using the patch clamp method. Asthe subject of the assay, Ca²⁺ dependant BigK channel (Kawakubo et. Am JPhysiol, 276 : H1827.1999) derived from heart muscle was employed. Byapplying this channel to the expressed ventricle muscle of chicken or toCHO cells force-expressing the cDNA of this channel with thecell-attached patch clamp method, an inside-out excised patch wasformed, and single channel current recordings were taken under a fixedmembrane potential. Assuming that the designed synthetic peptide blocksthe channel extracellularly the same as GsMTx-4, the space a fixeddistance above the glass pipette used for recording was filled with thepeptide of a known concentration beforehand, and administration wascarried out by back-fill which utilizes diffusion to reach the channel,With this method, the concentration of the peptide inside the pipettereaches equilibrium 15 to 20 minutes after the start of diffusion, andso the dissociation constant of the peptide can be estimated from theinhibition ratio at 20 minutes and onwards. Or, the relative inhibitionof the peptide can be estimated from the time taken for inhibition. Ifthe exact dissociation constant of the peptide is to be calculated, itis necessary to prepare an outside-out excised patch, take singlechannel current recordings, analyze the inhibition effects of variouspeptides with different concentration, and obtain a dose-inhibitioncurve; however, as this method requires special technology and as thisis the primary screening of the various designed synthetic peptides, theassay method combining the inside-out excised patch and back-filldescribed above was used to make a rough estimate of the inhibitioneffects of the peptides. Taking into consideration the results obtainedfor GsMTx-4, the concentration of the peptide was maintained at 10 μM.Concerning the scale of evaluation, the channel's open probability (Po,indicated in percentage) was used, and the level of inhibition wasexpressed by the inhibition ratio (percentage) with the control (priorto the administration of the peptide) as the standard, or by the timetaken for inhibition.

(Results of the Assay)

Of the five designed synthetic peptides, TVP003, TVP004 and TVP005displayed inhibiting activity. Therefore, mutation TVP017 (SEQ ID NO:16)in which the 3^(rd) R from the N-terminus of TVP004 had been substitutedby A and mutation TVP019 in which the 7^(th) K from the N-terminus ofTVP004 had been substituted by A were synthesized, and the inhibitingactivity was measured using the bioassay described above; as a result,these synthetic peptides were also found to show inhibiting activity.

The inhibiting activity of TVP003 is shown in FIG. 8. FIG. 8( a) is arepresentation of single channel current recordings. FIG. 8( b)represents the channel's open probability (Po). As indicated in FIG. 8,TVP003 showed 100% inhibition after 8 minutes, and so the dissociationconstant of TVP003 was estimated as μM order or lower. It is suggestedfrom this value that TVP003 shows inhibiting activity of a level thesame as or higher than the natural peptide from spider venom, GsMTx-4.

The inhibiting activity of TVP004 is shown in FIG. 9. FIG. 9( a) is arepresentation of the single channel recordings. FIG. 9( b) representsthe channel's open probability (Po) As indicated in FIG. 9, at 10 μM,TVP004 showed inhibition activity of about 60% after 8 minutes, and 95%after 16 minutes, displaying a relatively high level of inhibitionactivity.

The inhibition activity of TVP005 is shown in FIG. 10. FIG. 10( a) is arepresentation-of the single channel recordings. FIG. 10( b) representsthe channel's open probability (Po). As indicated in FIG. 10, TVP005showed about 60% inhibition after 20 minutes, and so the dissociationconstant thereof was estimated as approximately about 10 μM.

The inhibition activity of TVP017 is shown in FIG. 11. FIG. 11( a) is arepresentation of the single channel recordings. FIG. 11( b) representsthe channel's open probability (Po). As indicated in FIG. 11, TVP017showed 100% inhibition after 6 minutes, and so was found to display aneven higher level of inhibition than TVP003. It is suggested from thisvalue that TVP017 shows inhibition activity of a higher level than thenatural peptide from spider venom, GsMTx-4.

The inhibition activity of TVP019 is shown in FIG. 12. FIG. 12( a) is arepresentation of the single channel recordings. FIG. 12( b) representsthe channel's open probability (Po). As indicated in FIG. 12, TVP019showed approximately 80% inhibition after 14 minutes.

Embodiment 2: Specificity of the Peptide to the Channel Example 4:Specificity of the Active Peptide to a Mechano-Sensitve Channel

The specificity of the peptide of the present invention to amechano-sensitive channel was examined. TVP003 was used as the activepeptide, and myocardial SA channel, the same as that utilized in example3, was used as the channel. This channel has a STREX sequence consistingof 59 amino acids at the C-terminus, and the STREX-deletion-mutantdeleted of that sequence loses almost all extension activity, thusbecoming a common Ca dependant bigK channel (SAKCA: Tang, Naruse,Sokabe, J Membr Biol, 169:185-200, 2003). FIG. 13 is the result of studyinto the specificity of the active peptide to a mechano-sensitivechannel. FIG. 13( a) is a representation of the single channelrecordings related to the myocardial SA channel of TVP003. FIG. 13( b)is a representation of the single channel recordings related to theSTREX-deletion-mutant of TVP003. FIG. 13( c) represents the channel'sopen probability (Po). It can be seen from FIG. 13 that GsMTx-4 andTVP003 inhibit the wild-type SA channel but do not inhibit theSTREX-deletion-mutant hardly at all. Therefore, GsMTx-4 and TVP003 canbe considered as specifically acting only on a channel with extensionactivity.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to obtain a novelpolypeptide that specifically inhibits the activity of amechano-sensitive channel.

Utilizing a polynucleotide encoding such a polypeptide of the presentinvention, a recombinant vector comprising this polynucleotide, and atransformant transformed by the recombinant vector, the polypeptide ofthe present invention can be produced in large quantities.

A mechano-sensitive channel inhibitor comprising the polypeptide of thepresent invention or salts of the polypeptide of the present inventionis useful for manufacturing a reagent related to a mechano-sensitivechannel.

A remedy for atrial fibrillation comprising the polypeptide of thepresent invention or salts of the polypeptide of the present inventioncan efficiently treat atrial fibrillation.

1. A purified polypeptide or salt thereof, the polypeptide consistingof: an amino acid of SEQ ID NO: 2 an amino acid of SEQ ID NO: 16 or anamino acid of SEQ ID NO:
 17. 2. The polypeptide or salts in accordancewith claim 1, wherein the polypeptide having an intermolecular disulfidebond between two of the cysteines contained in the amino acid of SEQ IDNO: 2, the amino acid of SEQ ID NO: 16 or the amino acid of SEQ ID NO:17.
 3. A purified polypeptide or salts thereof, the polypeptideconsisting of: an amino acid of SEQ ID NO: 2 of which one or two aminoacids have been deleted, one or two amino acids have been substituted,one or two amino acids have been inserted or one or two amino acids havebeen added; the polypeptide having an intermolecular disulfide bondbetween two of the cysteines; and the polypeptide havingmechano-sensitive channel inhibiting activity.
 4. A mechano-sensitivechannel inhibitor comprising the purified polypeptide or salt thereof asdescribed in claim
 1. 5. A mechano-sensitive channel inhibitorcomprising the purified polypeptide or salt thereof as described inclaim
 2. 6. A mechano-sensitive channel inhibitor comprising thepurified polypeptide or salt thereof as described in claim
 3. 7. Aremedy of atrial fibrillation comprising the step of administering oneof the purified polypeptide or salts thereof described in claim
 1. 8. Aremedy of atrial fibrillation comprising the step of administering oneof the purified polypeptide or salts thereof described in claim
 2. 9. Aremedy of atrial fibrillation comprising the step of administering oneof the purified polypeptide or salts thereof described in claim
 3. 10. Apolynucleotide consisting of the polynucleotide that encodes thepolypeptide described in claim
 1. 11. A polynucleotide consisting of thepolynucleotide that encodes the polypeptide described in claim
 2. 12. Apolynucleotide consisting of the polynucleotide that encodes thepolypeptide described in claim
 3. 13. A recombinant vector comprisingthe polynucleotide described in claim
 10. 14. A recombinant vectorcomprising the polynucleotide described in claim
 11. 15. A recombinantvector comprising the polynucleotide described in claim
 12. 16. Atransformant transformed with the recombinant vector described in claim10.
 17. A transformant transformed with the recombinant vector describedin claim
 11. 18. A transformant transformed with the recombinant vectordescribed in claim 12.